Protein crystallization in microfluidic structures

ABSTRACT

A device for promoting protein crystal growth (PCG) using microfluidic channels. A protein sample and a solvent solution are combined within a microfluidic channel having laminar flow characteristics which forms diffusion zones, providing for a well defined crystallization. Protein crystals can then be harvested from the device. The device is particularly suited for microgravity conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application takes priority from U.S. ProvisionalApplication Serial No. 60/193,867, filed Mar. 31, 2000, whichapplication is incorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to a device for growingcrystals, and, more particularly, to a device for promoting proteincrystal growth using microfluidic structures.

[0004] 2. Description of the Related Art

[0005] Macromolecular crystals have become keystones of molecularbiology, biochemistry, and biotechnology. Understanding how crystalsexpress their function depends on knowledge of the macromoleculararchitecture at the atomic level.

[0006] The determination of the three dimensional atomic structure ofcrystals is one of the most important areas of pure and appliedresearch. This field, known as X-ray crystallography, utilizes thediffraction of X-rays from crystals in order to determine the precisearrangement of atoms within the crystal. The result may reveal theatomic structure of substances as varied as metal alloys to thestructure of deoxyribonucleic acid (DNA). The limiting step in all ofthese areas of research involves the growth of a suitable crystallinesample.

[0007] One important and rapidly growing field of crystallography isprotein crystallography. Proteins are polymers of amino acids andcontain thousands of atoms in each molecule. Considering that there are20 essential amino acids in nature, one can see that there existsvirtually an inexhaustible number of combinations of amino acids to formprotein molecules. Inherent in the amino acid sequence or primarystructure is the information necessary to predict the three dimensionalstructure. Unfortunately, science has not yet progressed to the levelwhere this information can be obtained quickly and easily. Althoughconsiderable advances are being made in the area of high field nuclearmagnetic resonance, at the present time the only method capable ofproducing a highly accurate three dimensional structure of a protein isby the application of X-ray crystallography. This requires the growth ofreasonably ordered protein crystals (crystals which diffract X-rays toat least 3.0 angstroms resolution or less), as the accuracy ofstructures determined by X-ray crystallography is limited by thedisorder in the crystallized protein.

[0008] The maximum extent of a diffraction pattern is generallyconsidered to be a function of the inherent statistical disorder of themolecules of protein crystals rather than the result of purely thermaleffects. Statistical disorder present in protein crystals has twoprincipal sources: 1) intrinsic structural or conformational variabilityof protein molecules, and 2) spatial distribution of the individualmolecules about lattice sites occupied.

[0009] In addition, other inherent limitations in the crystallizationprocess involve the effects of molecular convection, thermal effects,and buoyancy, all due to the earth's gravitational field. Therefore, ithas been proposed to conduct crystallization experiments in themicrogravity ({fraction (1/1000)} g to {fraction (1/10,000)} g) ofspace, on board the space shuttle, international space station, or othersimilar vehicles. Several patents disclose crystallization inmicrogravity to improve the size, morphology and diffraction quality ofcrystals. U.S. Pat. Nos. 5,362,325 and 4,755,363 are exemplary ofpatents disclosing microgravity crystallization.

[0010] Focus of microgravity research in protein crystal growth (PCG)has been based on the observation that PCG in a microgravity environmentyields protein crystals that are of reduced disorder. Reduction inlattice disorder by protein crystals grown in microgravity compared toground controls offers enhanced resolution of diffracted intensities andtranslates at the atomic level into more precise knowledge of theprotein architecture. The detailed knowledge of how ligands interactwith binding sites at the atomic level permits insight into catalyticmechanisms and recognition in biological systems, a prerequisite forstructure-assisted drug design. In a pharmaceutical industry setting,higher resolution implies significant manpower reduction in syntheticchemistry to explore the drug-binding site and results in more rapidoptimization of drug target interaction. Accelerated drug design isextremely cost effective, allowing a pharmaceutical company to quicklyrecover R&D costs and improve profitability.

[0011] Several important advances have recently accelerated thestructure determination process using even small crystals. These includeselenomethionyl proteins, cryo-crystallography, high intensitysynchrotron radiation sources, CCD detectors, and multiwavelengthanomalous diffraction (MAD) phasing. With these advances, a proteinstructure can be solved by MAD phasing literally within hours of datacollection at a synchrotron radiation source. The outstandinguncertainty faced by protein crystallography is the growth of highquality protein crystals.

[0012] In the very near future, it is expected that the field ofstructural genomics will foster a tremendous explosion in demand forprotein structure determination. Genome sequencing or genomics issignificantly impacting biological research by changing ourunderstanding of biological processes through identification of novelproteins that may be involved in disease or are unique to pathogenicorganisms. Genome project results have shown that in most organisms,more than 50% of the proteins have no assigned function. In the humangenome, this amounts to over 50,000 proteins. These uncharacterizedproteins thus represent a reservoir of untapped biological informationthat is widely acknowledged as the next generation of proteintherapeutics and targets for pharmaceutical development. Withlarge-scale genomic sequencing now becoming routine, attention is beingfocused on understanding the structure and function of these biologicalmacromolecules. Recently published examples where knowledge of athree-dimensional structure of an unknown protein can provide clues toits function is expected to open the gates to a massive need for highquality structure determination.

[0013] The crystallization process generally involves several distinctphases, such as nucleation and post-nucleation growth. Nucleation is theinitial formation of an ordered grouping of a few protein molecules,while post-nucleation growth consists of the addition of proteinmolecules to the growing faces of the crystal lattice and requires lowerconcentrations than the nucleation phase.

[0014] Most protein crystals nucleate at very high levels ofsupersaturation, typically reaching up to 1000% in many cases. At suchsupersaturation levels, post-nucleation crystal growth takes place undervery unfavorable conditions. Most macromolecules at the concentrationsneeded to attain the very high levels of supersaturation tend to formaggregates and clusters of both ordered oligomeric species and/or randomamorphous aggregates. Depending on the half-life and concentration ofsuch clusters, formation of nuclei can involve incorporation ofpartially ordered aggregated species. Quiescent conditions mitigateimperfect post-nucleation growth at high supersaturation by reducing thecollision frequency of aggregate species of all kinds to form largerclusters or nuclei. Microseeding a protein solution, that is,introduction of freshly crushed crystallites, would provide a succinctapproach to circumvent growth from imperfect nuclei.

[0015] At higher levels of supersaturation, growth by absorption ofthree-dimensional nuclei onto crystal faces has been observed incrystallization studies of thaumatin, catalase, t-RNA, lysozyme, lipase,STMV virus and canavalin. The three-dimensional nuclei have observedaverage dimensions ranging between 1-10 μm making them colloidal insize. The origin of these nuclei is thought to be protein clusters thatoriginate from protein rich droplets possessing short-range internalorder and that undergo long-range ordering upon interaction with theunderlying crystal lattice.

[0016] Under quiescent conditions at low supersaturation, a proteincrystal grows by incorporation of individual protein molecules,monomers, from the surrounding medium, which because of their lowdiffusivities produce a concentration gradient or depletion zone aboutthe growing crystallite. For lysozyme, protein concentration gradientsmeasured by Mach-Zender interferometry surrounding a large 1 mm crystalare the order of ˜10% over a 2-3 mm distance. Larger aggregates in thebulk solution diffuse more slowly than protein monomers, allowing thedepletion zone to kinetically discriminate against incorporation oflarge aggregates into the crystal lattice. In effect, the depletion zoneacts much like a mass filter. The depletion zone not only tends tofilter out larger aggregates but also partially unfolded or denaturedproteins which also have larger hydrodynamic radii, hence lowerdiffusivities than the compact globular native protein. Since massfiltering is transient and based on differential diffusion of thevarious species, protein crystal growth will eventually be compromisedby self-impurities as the system approaches equilibrium. AFM studies inground controls have shown that macromolecular crystals tend to stopgrowing because of formation of a dense impurity adsorption layer ofprotein restricting access to crystal faces.

[0017] In microgravity, sedimentation and buoyancy convection effectsare suppressed and diffusion is the dominant mechanism of proteintransport. Hence, a depletion zone would be extended and could moreeffectively exclude higher order protein aggregates from incorporationinto a growing protein crystal, thus leading to a greater degree incrystal perfection. Recent PCG studies in microgravity with lysozymedimer self-impurities tend to support this hypothesis. Non-quiescentconditions such as gravity induced sedimentation of larger nuclei and/orcrystallites adjacent to the growing crystal would create disturbancesin the depletion zone, facilitating incorporation of higherconcentrations of self-impurities, and compromise its role of massfiltering. Post-nucleation growth by absorption of three-dimensionalnuclei, observed at higher supersaturation levels, is particularlysusceptible to sedimentation effects and buoyancy-driven flow. Particlessuch as nuclei of colloidal size are susceptible to gravitationaleffects and this may be in large part the basis for the beneficialeffect of microgravity on PCG.

[0018] Frequently, prior to activation of a PCG experiment inmicrogravity, purified protein is stored at high concentration for aslong as several weeks. For a protein maintained in soluble state,protein instability or unfolding promotes production of irreversibleaggregates. Thus, given the high supersaturation conditions required fornucleation, protein crystal nuclei may contain significantconcentrations of amorphous aggregates. Whether presence ofself-impurities is detrimental to subsequent ordered post-nucleationgrowth and hence crystal quality is a function of the ability ofcompetent nuclei to promote post-nucleation growth and concentration ofcompetent monomeric species. Clearly, highly ordered nuclei tend to bekinetically more stable than amorphous aggregates, which is essentialfor sustaining post-nucleation growth. However, under prolonged solutionstorage, irreversible protein aggregation may compromise PCG success.

[0019] Several methods of protein crystallization have been developedand successfully employed over the course of the last century. Theseinclude vapor phase diffusion, liquid-liquid interfacial diffusion,liquid-liquid turbulent mixing, and step gradient methods.

[0020] Approximately 90% of protein crystallization experiments in,microgravity (and on the ground) in the past decade have used the vapordiffusion or hanging drop method, in which water is transported throughthe vapor phase from a drop of protein and precipitant solution to aconcentrated precipitant solution. This method has several advantages,especially at 1×g, including the relative absence of container surfaces,slow approach to supersaturation, low volume requirement, and ease ofobservation of crystal nucleation and growth, and it is fairly viscosityindependent. It also has a number of disadvantages, including limitedvolume in the case of hanging (but not sessile) drops, limited controlover saturation rate, and a potential for the establishment ofconvection currents at the liquid—air interface. The sessile-hangingdrop, like the hanging-drop method, removes water only from thecrystal-growth solutions. Unlike hanging drop in the sessile dropmethod, buoyancy-driven fluid upwelling often occurs, and the rate ofwater removal depends on vapor pressure. Examples of devices which usethe vapor-diffusion method include U.S. Pat. Nos. 4,886,646; 5,103,531;5,096,676; and 5,130,105.

[0021] Interfacial diffusion or liquid-liquid interfacial diffusion as atechnique for protein crystal growth involves superposition of proteinand precipitant solutions across an interface. PCG then depends onmutual self-diffusion of protein and precipitant across the resultantinterface to grow protein crystals. Due to convection effects, suchinterfaces are not stable on earth but can be reproducibly generated inmicrogravity. The transient concentration gradient affords control overnucleation events by spatially reducing the number of nucleation sites.Protein dilution by the precipitant solution as system equilibrationtakes place diminishes the potential for protein aggregate incorporationinto nuclei and crystallites. In this method, a depletion zone will onlybe established once the system has approached equilibrium. Mixing ofhighly viscous fluids by interfacial diffusion occurs very slowly andcan correspond to a time scale incompatible with the duration of ashuttle mission but is compatible with the ISS mission.

[0022] Turbulent mixing swill result essentially in the system beingbrought to its equilibrium value at the onset of the PCG experiment andmaintained at equilibrium throughout the experiment. This is useful inallowing comparisons to be made where it is important to know the finalend point of a system and is akin to batch crystallization. Turbulentmixing also overcomes difficulties associated with mixing of viscousprecipitants.

[0023] In the step gradient approach, homogeneous nucleation and crystalgrowth are treated as separate steps. Homogeneous nucleation is inducedby bringing, carefully, a near saturated protein solution into contactwith a highly supersaturating solution of precipitant (1.2-3.0 timessaturating concentration, for example). This exposure lasts just longenough to cause nucleation, then the crystals are transferred to aslightly saturating concentration of precipitant for quiescent crystalgrowth. This method has been successfully used to grow protein crystalsin space.

[0024] An essential difference between vapor phase diffusion andliquid-liquid interfacial diffusion is in their mutually orthogonalapproach to equilibrium in the protein solubility phase. Vapor diffusionstarts from a dilute protein solution that becomes concentrated atequilibrium, while liquid-liquid interfacial diffusion dilutes theprotein starting condition.

[0025] All of the methods discussed above have gravity-dependentcomponents. Crystals more dense than the mother liquor sediment awayfrom the zone of crystallization, while those less dense float away fromthis zone. Sedimentation against a vessel wall modifies the habit of thecrystal. Rapid nucleation on a dialysis membrane or vessel wallsometimes leads to large numbers of small crystals. Ideally, motionless,contactless crystal growth is desired, and the microgravity environmentof space flight comes very close to providing these conditions.

[0026] Modern protein crystallography data collection techniques makeuse of protein crystals flash frozen in liquid nitrogen to minimizeradiation damage. Crystals of large dimensions (0.5-1 mm) are morereadily damaged during flash freezing while smaller crystals (˜0.2 mm orless in average dimension) can be cooled rapidly enough to prevent iceformation. Using 2^(nd) and 3^(rd) generation synchrotron radiationsources and CCD detectors, even smaller crystals have been successfullyexploited. The device of the present invention thus targets growth ofhigh quality small and medium size crystals. The presence ofself-impurities is more likely to compromise growth of larger crystalsthan smaller crystals largely in part to the longer time scale involvedfor growth of large crystals, making them more susceptible to proteindenaturation phenomena.

[0027] Protein denaturation, if it does occur prior to PCG activation inmicrogravity, can compromise PCG success by formation of irreversibleaggregates, self-impurities, in the protein solution. If irreversibleaggregation does take place in ground experiments and compromises PCGsuccess, the facility should be able to mitigate against the proteinaggregate population at the time of PCG activation.

[0028] Protein crystals can be stressed or even damaged duringharvesting and/or in subsequent manipulations and therefore becomeunsuitable for data collection. The present device should allow facileharvesting of protein crystals for flash freezing. In particular,potential crystal entrapment in corners should be avoided.

[0029] The device should afford facile integration and dispersement oflarge number of PCG experiments by a PCG mission integration center aswell as allow ready documentation of post-flight results.

[0030] The PCG experiment should allow for crystallization in smallvolumes comparable to volumes (μL) used in routine laboratory PCGscreening, thus consuming as little protein as possible.

[0031] Technically, the facility should provide efficient separation ofprotein and precipitant prior to orbit activation with no absorption andleakage of fluids over the course of the microgravity mission.

[0032] Microfluidic devices have been recognized to have great potentialin such areas as DNA sequencing and medical diagnostics. Beyond this,they have the potential to allow separations, chemical reactions, andcalibration-free analytical measurements to be performed directly onvery small quantities of complex samples such as whole blood andcontaminated environmental samples. Therefore, use of disposablemicrofluidic devices should be investigated as means for growing proteincrystals in microgravity.

[0033] The embodiment of the technology uses microfluidic integratedcircuits. These devices are thin transparent plastic or glassstructures, roughly credit card in size. Laminar flow structures inthese chips afford crystal growth by free liquid-liquid interfacediffusion, batch methods, or vapor diffusion, depending on circuitdesign. The chips are readily loaded with fluid samples, which,manufactured from transparent material, allow facile documentation ofPCG results and also permit facile unloading and harvesting of proteincrystals grown.

[0034] Most fluids show laminar behavior in miniature flow structureswith channel cross sections below 0.5 mm. Two or more distinct fluidstreams moving in such flow structures do not develop turbulence at theinterface between them or at the interface with the capillary walls.Different layers of miscible fluids and particles can thus flow next toeach other in a microchannel without any interaction, other than bydiffusion of their constituent molecular and particulate components.Microfluidic channels typically have either width or height less than˜500 μm. Liquids with viscosities comparable to water or that flowslower than several cm/sec follow predictable laminar paths. Theseconditions correspond to values of the non-dimensional Reynolds numbersof ˜1 or less. The Reynolds number characterizes the tendency of aflowing liquid to develop turbulence; values greater than 2000 indicateturbulent flow. Values between 1 and 2000 allow for so-called laminarrecirculation, which is frequently used in microfluidic mixingstructures.

[0035] Recent advances in device miniaturization have led to thedevelopment of integrated microfluidic devices, so-calledlabs-on-a-chip. In these tiny microchips etched with grooves andchambers, a multitude of chemical and physical processes for bothchemical analysis and synthesis can occur. These devices, also known asmicro-total analysis systems (μTAS), can be mass produced in silicon bytechniques similar to those used in the semiconductor industry, or, foreven lower cost, made out of plastics by using casting, cutting, andstamping techniques. Recent advances in microfabrication have extendedthe production of these devices to include a wide range of materials.They offer many advantages over traditional analytical devices: theyconsume extremely low volumes of both samples and reagents. Each chip isinexpensive and small. The sampling-to-result time is extremely short.In addition, because of the unique characteristics exhibited by fluidsflowing in microchannels (“microfluidics”), it is possible that thesedesigns of analytical devices and assay formats would not function on amacroscale. For PCG, microfluidic structures offer a novel, innovativeand modular concept different from the current available PCG hardware.There are a number of ways in which these microfluidic structures arerelevant to PCG.

[0036] Several microfluidic structures have been recently developedwhich can be useful as “building blocks” for a variety of differentdisposable crystallization chips. These devices make it possible todeliver small volumes (tens of nanoliters to tens of microliters) ofsample and reagents at flow rates down to nanoliters per second.

[0037] Due to the low Reynolds Number conditions in microfluidicsystems, mixing is usually limited to laminar diffusion mixing orlaminar recirculating mixing. However, it is possible to introduceturbulence into microfluidic systems. Devices have been developed whichallow quasi-turbulent mixing of both two or more single-phase liquids orliquids containing solid particles. It consists of a series of chambers,connected by small-diameter channels. Once the mixer is filled, thefluid contained in the mixer can be subjected to a series of reversalsof direction. Each time the fluid is pulsed in the forward or reversedirection, each tangential channel produces a laminar jet in eachchamber. Because each laminar jet causes the fluid in each chamber torotate as a vortex in the same direction, the rotational shear fieldinduces mixing. Fluid mixing can also be achieved by separately dividingeach fluid channel into narrow finger channels and then recombining theall finger channels into one channel.

[0038] U.S. Pat. Nos. 5,716,852 and 5,932,100 are directed tomicrofluidic structures which operate on the principle of laminar flowwithin a microscale channel wherein separate input streams are placed inlaminar contact within a single flow channel such that desired particlescan be detected or extracted by virtue of diffusion. U.S. Pat. No.5,716,852, which patent is hereby incorporated by reference, discloses adevice, known as a T-Sensor, which can be used to analyze the presenceand concentration of small particles in streams containing both smallparticles and larger ones by diffusion principles. The speed of thediffusion mixing is a function of the size of the diffusion particles.U.S. Pat. No. 5,932,100, which also is hereby incorporated by reference,discloses a device known as an H-Filter, which, by laminar flow, allowsseparation of particles based on diffusion coefficients on a continuousbasis without the need for semipermeable membranes. The H-Filter canalso be used as a dilution tool, or, by using several H-Filters inseries, a highly accurate serial dilution structure.

[0039] Although not directly related to the concept study, understandingof a T-Sensor operation is necessary for appreciation of the PCG conceptdesign. A T-Sensor is a micro-total analysis system (μTAS) componentthat combines the separation features of the H-Filter with detection. AT-Sensor system is demonstrated in which a sample solution, an indicatorsolution, and a reference solution are introduced in a common channel.The fluids interact during parallel flow until they exit themicrostructure. Large particles such as blood cells would not diffusesignificantly within the time the flow streams are in contact. Smallatoms such as H⁺, Na⁺, and small molecules diffuse rapidly betweenstreams, whereas larger polymers diffuse more slowly and equilibratebetween streams further from the point of entry to the device. Asinterdiffusion proceeds, interaction zones are formed in which sampleand reagents may bind and react. T-Sensors can be used to let componentsfrom two different, but miscible streams diffuse into one another andreact with each other. For example, antigens contained in one stream candiffuse into a parallel stream containing antigens, and react with them,while the two original streams remain largely separate.

[0040] T-Sensor-like structures can be used to induce precipitation orcrystallization of sample components. For example, components from onestream can diffuse into a parallel stream and react with a componentthere to form a precipitate. Alternatively, solvent molecules from onestream can diffuse into a parallel stream containing a different solventand particles. The change in solvent properties along the diffusioninterface zone can then induce crystallization or precipitation.Obviously, it is also possible to apply a temperature gradient to amicrochannel, either across the channel or along its flow direction, andaffect the precipitation characteristics this way. Microseeding would beanother possibility with this device.

[0041] It should be noted that it is possible to mitigate againstprotein instability using microfluidic technology. Protein denaturationresults in polydisperse protein populations that contain higher orderprotein aggregates. The concentration of these aggregates can beminimized or even eliminated through use of an H-filter structurebecause of the difference in diffusion coefficients between nativeprotein and protein aggregates. An H-filter set up, would preferentiallyconcentrate the monodisperse native protein in the filter output.

[0042] Another microfluidic device which may be useful with respect toPCG is described in U.S. Pat. No. 5,726,751, which patent is herebyincorporated by reference. This patent discloses a device, known as amicrocytometer, which is based on a sheath flow cytometer design, andhas at its heart a disposable laminate cartridge technology developedspecifically for microfluidic devices. It may be possible using thistechnology to focus precipitating crystals using a combination ofmicrofluidic hydrodynamic and geometric focusing structures. This wouldline up the particles in a single file as they flow past a detector, orallow them to settle out on the bottom of the structure in a verycontrolled and precise way.

[0043] Finally, several other devices which were developed in view ofmicrofluidic technology are taught in U.S. Pat. Nos. 5,474,349;5,726,404; 5,971,158; 5,974,867; 6,007,775; 5,948,684; and 5,922,210;these patents are also hereby incorporated by reference into the presentapplication.

SUMMARY OF THE INVENTION

[0044] Accordingly, it is an object of the present invention to providea device for growing protein crystals using microfluidic structures.

[0045] It is also an object of the present invention to provide a devicein which multiple assays can be performed simultaneously.

[0046] It is a further object of the present invention to provide adevice in which small volumes of liquids can be used to perform proteincrystal growth (PCG) experimentation.

[0047] It is a still further object of the present invention to providea device which is easy to use under microgravity conditions.

[0048] These and other objects and advantages of the present inventionwill be readily apparent in the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049]FIG. 1 is a graphic representation of a T-Sensor which may be usedin the present invention;

[0050]FIG. 2 is a graphic representation of the T-Sensor of FIG. 1 inwhich the two input fluids are premixed;

[0051]FIG. 3 is a graphic representation of the T-Sensor of FIG. 2 whichsimulates the vapor phase or hanging drop diffusion method of proteincrystallization;

[0052]FIGS. 4A and B show graphic representations of several moleculeswhich have been mixed in a diffusion mixer after an elapsed time period.

[0053]FIG. 5 is a top view of a microfluidic cartridge for use in thepresent invention shown in the loading mode;

[0054]FIG. 6 is a top view of the cartridge of FIG. 5 shown in theactivation mode;

[0055]FIG. 7 is a top view showing the loading mode of a microfluidiccartridge showing another embodiment for carrying out the presentinvention.

[0056]FIG. 8 is a top view showing the loading mode of a microfluidiccartridge showing another embodiment for carrying out the presentinvention;

[0057]FIG. 9 is a top view showing the loading mode of a microfluidiccartridge showing another embodiment for carrying out the presentinvention; and

[0058]FIG. 10 is a top view showing the loading mode of a microfluidiccartridge showing another embodiment for carrying out the presentinvention.

[0059]FIG. 11 is a top view showing the loading mode of a microfluidiccartridge for performing high density screening crystallization.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] Solution conditions that promote ordered protein aggregation arefavorable for protein crystallization. Aggregation involves proteininteractions mediated through specific forces that are sensitive toprotein surface topology and chemical identity of the surface groups.The complexity of these interactions represent the difficultyencountered in obtaining X-ray diffraction quality crystals. Whiledeveloped for very simple particle interactions, statistical mechanicalmodels of order/disorder phase transitions have offered insights intohow to characterize the effect of solution conditions on solubility.Attempts to characterize proteins as simple fluids suggest that, undermost crystallization conditions, proteins experience attractions, whichhave a range much shorter than their size. This has importantconsequences on protein solution phase behavior. First, the solubilityat a given strength of attraction becomes weakly dependent on the extentof the attraction. Consequently, large classes of proteins can display anarrow range of solubility at a given level of attraction and on whichprotein crystallization is dependent.

[0061] One method of characterizing the strength of the attraction is tomeasure the protein 2^(nd) virial coefficient. A second consequence ofthe short-range nature of the interaction potential is that proteinsolutions will show through density fluctuations a metastablefluid/fluid phase transition. This transition appears as a phaseseparation into two solutions: one rich in protein and one dilute inprotein. The critical point for this phase transition lies at strongerattractions than the fluid/crystal phase boundary. Therefore, crystalswill ultimately grow from the protein rich phase-separated state. Theproximity of the critical point to the fluid/crystal phase boundaryplays an important role in crystal nucleation and which is linked to thenarrow range of the protein 2^(nd) virial coefficient values consistentwith protein crystallization. The role of additives or differentstarting conditions is to modify fluid/fluid phase boundaries and createsolution conditions favorable for protein crystallization.

[0062] Interaction between protein molecules is concentration dependentand can be assayed from light scattering measurements. The dependence ofa light scattering on protein concentration in dilute protein solutionsis directly informative as to the extent of protein interaction and theconstant characterizing this dependence is the 2^(nd) virialcoefficient, B₂. Positive values for B₂ are qualitatively representativeof repulsion between protein molecules while negative values indicateattractive interactions between protein molecules. Large negative valuesof B₂ imply strong attractions between protein molecules that result ingel formation or amorphous precipitation. George and Wilson observedthat there was a commonality to the solution conditions that arefavorable for protein crystallization, and that commonality could beexpressed by the 2^(nd) virial coefficient, B₂. The measured values forB₂ using many different protein-solvent pairs all, unambiguously, fallinto a fairly narrow range referred to as the crystallization slot. Thisslot is an empirical representation of solution conditions for which PCGwas successful. The B₂ values comprising the slot are slightly tomoderately negative (≈−1 to −8×10⁻⁴ mol ml g⁻²) and represent slightlyto moderately net attractive forces between protein molecules.

[0063] Static light scattering (SLS) is the analytical method used todetermine the 2^(nd) virial coefficient, B₂. This method requires theintensity of light scattered by a protein solution in excess ofbackground due to solvent and stray light to be measured as a functionof the protein concentration. The working relationship used to analyzethe SLS data is given by the following equation:$\frac{K\quad c}{R_{\theta}} = {\frac{1}{M} + {2B_{2}c} + \ldots}$

[0064] where K is an optical constant dependent on refractive index,Avogadro number, wavelength of incident light and change of refractiveindex with protein concentration c. The excess Rayleigh ratio, R_(θ),measured at a scattering angle of θ is determined as a function ofprotein concentration c. M represents the molecular weight. By plottingKc/R_(θ) versus c, the 2^(nd) virial coefficient, B₂, can be obtainedfrom the limiting slope. B₂ is a dilute solution parameter and theprotein concentration used for the SLS data depends on detectionsensitivity, ranges are typically 0.05 mg/ml for proteins of largemolecular weight to 1 mg/ml for lysozyme. In comparison to proteinconcentrations used for PCG, the 2^(nd) virial coefficient can bedetermined using small quantities of protein.

[0065] Surfactants are required to solubilize membrane proteins.Therefore, in order to crystallize a membrane protein one mustcrystallize the complex of protein bound to the surfactant used. Mostmembrane protein crystals to date have been observed to form near thecloud point of the surfactant used. This cloud point is the surfactantphase separation boundary corresponding to the aggregation of surfactantmicelles; as a solution approaches the cloudpoint, intermicellarpotentials switch from repulsive to attractive. As static lightscattering is sensitive to micellar structures, determination of thesecond osmotic virial coefficient (B₂) for the protein-surfactantcomplex must take into account the interactions between scatteringmicelles. A T-sensor detection structure described below allowsmeasurement of the second osmotic virial coefficient for theprotein-surfactant complex in presence of the surfactant micelles.

[0066] Although the value of the 2^(nd) virial coefficient is predictiveof crystallization conditions, not all starting condition consistentwith a crystallization slot value for the 2^(nd) virial coefficientguarantee diffraction quality crystals. Hence the value of 2^(nd) virialcoefficient will be used to filter starting conditions, conducive forPCG trials, and all conditions will be screened that correspond toweakly attractive protein interactions that bracket the B₂crystallization slot value.

[0067] Two general types of “gravity-driven” microfluidic structureshave been manufactured: the “vertical” (GVT and GVH) types, which haveintegrated sample and reagent reservoirs, and which are operatedvertically or at an incline, and the “horizontal” (GHT and GHH) types,which have tubes attached to them for sample and reagent filling. Theletter code stands for Gravity-driven Horizontal (or Vertical) H-Filter(or T-Sensor). H-Filters have two inlets and two outlets, are designedto separate components of a sample solution, and allow the collection ofthe output solutions. T-Sensors have two or three inlets, and only onewaste outlet. They allow the detection of analytes directly in complexsample solutions (such as whole blood). They are filled with a samplesolution, a indicator solution, and, for three inlet-T-Sensors, anadditional reference solution with a known concentration of analyte.

[0068] In both GH- and GV-type structures, the flow rate depends on thehydrostatic height of the flow column in each of the inlets, and each ofthe outlets. This means that the flow speed as well as the relativeposition of the centerline between the two streams can be adjusted bychanging the height of the fluid column in each inlet and outlet. Someof the T-Sensor types are less sensitive to differences in the fluidcolumn height; others are more sensitive, but these also allow to adjustthe centerline very accurately.

[0069] Both GVT and GHT types can be filled with the “filling syringes”.For GV-types, place the blunt needle inside the hole of the reservoirson top of each cartridge. It is easiest if the needle is placed somewhatto the side of the hole, and the cartridge is held at a slight downwardangle; fill slowly and carefully to avoid air bubbles. The reservoirdoes not need to be filled completely; however, the area close to thejunction with the inlet channels must be covered with fluid.

[0070] Sometimes the flow starts as soon as the liquid is placed in thetube; if it is required that the fluids do not mix at all before theyenter the main T-sensor channel, then all reservoirs should be filledwhile the GV cartridge lies flat. Filling GHT-type cartridges issomewhat easier; just fill sample and indicator into both inlet tubes atthe same time and to the same level using two syringes or pipettes.

[0071] For both GH and GV types, frequently the flow does not start byitself when the fluids are in the tube or the inlet reservoirs. In thiscase, place the “aspiration syringe” with the silicon tip (enclosed)over the outlet channel opening and aspirate slightly until the fluidsstart flowing from all inlets. Keep aspirating until all air bubblesthat may have formed are removed from the channels. Fluids should nowflow unaided as a function of hydrostatic pressure alone.

[0072] The flow speed can be adjusted by adding or removing fluid fromthe inlet tubes (GH types), or by adjusting the incline of thecartridges (GV-types). The higher the height difference between inletand outlet fluid levels, the faster the fluids will flow for a givenstructure. Alternatively, the flow can be increased by placing a Q-Tipon the outlet opening (once the fluid has reached the outlet), whichincreases the flow dramatically through absorptive action.

[0073] The following presents a description of certain specificembodiments of the present invention. However, the present invention canbe embodied in a multitude of different ways as defined and covered bythe claims. Throughout the drawings, like parts are designated with likeindex numerals throughout.

[0074] A T-Sensor-like structure, generally indicated at 10, is shown inFIG. 1 to demonstrate the principles of diffusion-based crystallization.A sample 12 containing dissolved protein, and a reagent 14 containing avariety of different solvents and salts, flow together in parallelwithin a channel 15 of T-Sensor 10. After establishing a laminar flowprofile, the flow is significantly slowed or stopped. The variouscomponents of both streams 12, 14 will now diffuse into each other at acertain rate, depending on the size of the molecules within thesestreams, forming diffusion interface zones 16, 18 within channel 15 ofdevice 10. This action establishes a concentration gradient in device10, which allows for a very well defined crystallization. Solventmolecules from one stream can diffuse into a parallel stream containinga different solvent and particles. The change in solvent propertiesalong diffusion interface zones 16, 18 can then induce crystallizationor precipitation. Obviously, it is also possible to apply a temperaturegradient to a microchannel, either across the channel or along its flowdirection, and affect the precipitation characteristics this way.Microseeding would be another possibility with this device.

[0075] Referring now to FIG. 2, a microfluidic rapid mixing structure20, such as a laminar jet vortex mixer which is described in U.S. patentSer. No. 60/206,878, a split-channel diffusion mixer, or any other mixerthat rapidly mixes fluids in the low Reynolds-number regime can beplaced upstream of crystallization channel 15. The protein sample andthe reagent are mixed at a certain ratio, and then flow intocrystallization channel 15, where a homogeneously mixed solution 22 isslowed or stopped. Crystallization will then occur inside channel 15.Again, microseeding or temperature gradients can also be applied.

[0076]FIGS. 4A and 4B show the behavior of two different molecules whenmixed using a diffusion mixer. The figures demonstrate that, withinabout 2 minutes, even large molecules are completely equilibrated across100-micrometer wide channels that make up the split-channel diffusionmixer. FIG. 4A shows a phosvitin complex (1,490,000 MW) concentration(Z) in a 100 μm channel (X) for 120 seconds (Y), while FIG. 4B shows athyrogobulin (bovine) (669,000 MW) concentration (Z) in a 100 μm channel(X) for 120 seconds (Y).

[0077] Referring now to FIG. 3, T-Sensor 10 of FIG. 2 is again used; butin this embodiment, crystallization channel 15 is filled only partially.Exit end of channel 15 is connected to an absorbing material 24 thatabsorbs, over time, a predefined quantity of solvent mixed solution from22, thereby increasing the concentration of protein, and inducing it tocrystallize. Again, microseeding or temperature gradients can also beapplied in this embodiment.

[0078] A prototype for 12 PCG experiments on a single card is shown intwo different operational modes in FIGS. 5 and 6. A single microfluidicPCG experiment embodies the following elements: a driver fluid interface30, two fluid reservoirs 32, 34 and microfluidic channel/check valves36, 38, crystallization chamber 39, harvesting chambers 40, microchannelconnections 42 and adhesive sealing means 44.

[0079] Referring now to FIG. 5, a microfluidic cartridge, generallyindicated at 50, contains a plurality of fluid reservoirs 32, 34.Reservoirs 32 are filled with a protein sample, while reservoirs 34 arefilled with a precipitant solution. Fluids in reservoirs 32, 34 areexpelled by applying pressure to a fluid located within channel 30,which may be air or an inert oil. Reservoirs 32, 34 combine to form aT-sensor structure with crystallization chamber 33. Laminar flow ensuresthat the two fluids do not mix within chamber 39 other than by mutualself-diffusion. The contents of crystallization chamber 39 void intoharvesting chamber 40. Each fluid reservoir 32, 34 is filled through afluid inlet 52 and has microfluidic channel/check valves 36, 38 a venthole 54 to permit air escape during the filling operation. Surfacetension effects because of the small diameter of the connecting to thefluid reservoirs 32, 34 prevent fluids flowing out of said reservoirs.Once loaded, fluid reservoirs 32, 34 are carefully sealed with adhesivestrip 44, as can be seen in FIG. 6. This strip 44 can be supplieddirectly bonded to cartridge 50. Harvesting chambers 40 are sealed withanother strip 44 of adhesive tape also supplied directly on cartridge50. In microgravity or for long-term storage prior to fluid activation,the check valves 36, 38 minimize vapor loss from reservoirs 32, 34.Check valves 36, 38 allow fluid flow in one direction only such thatback flow is prevented, and when appropriately placed within amicrofluidic circuit, can act as one level of fluid containment.

[0080] External valve activation and fluid driving can be accomplishedin one of two ways: using an external driver or by air bellowsincorporated on the microfluidic cartridge. An external fluid driverinterface 60 (FIG. 6) would be an air pump to which each card would behooked up. Air pump 60 delivers a precise amount of pressure to drivefluids through the circuit. Another option is to use an air bellows 62,as shown in FIG. 5, directly manufactured on the circuit board that canbe driven by pressure to pump the fluids into the microfluidicstructures. Air bellows 62 may also have a vent hole 64, which may besealed by a ball bearing, and when under pressure air bellows 62 wouldagain act as the fluid driver. Release of pressure due to sudden poweroutage would allow air to bleed into the microfluidic circuit, allowingit to equilibrate. Check valves 36, 38 in any event would prevent fluidback flow and satisfy one level of containment. The advantage of venthole 64 on the air bellows 62 is that circuit cartridge 50, onceactuated, could be allowed to slowly return to equilibrium and thenallow facile harvesting of crystal chamber 40 contents by applyinganother round of pressure on the bellows 62. It is also possible to fillbellows 62 with inert oil to drive the fluids and prevent vapor loss inthe microfluidic cartridge 50 over the long-term course of a PCGexperiment, if this becomes necessary.

[0081] Activation by applying pressure on the driver fluid withinchannel 30 by bellows 62 pumps the fluid reservoirs 32, 34 contents intocrystallization chamber 39 via check valves 36, 38. Check valves 38ensure that there is no back flow from crystallization chamber 39 whilecheck valves 36 ensure a further level of fluid containment. Harvestingof a particular PCG experiment occurs by partially peeling off theadhesive strip 44 to allow access to the chosen harvesting chamber 40.Circuit pressurization via fluid driver interface 60 or air bellows 62would allow flushing with inert oil or air of the crystallizationchamber 39 contents. Crystals are then accessible for facile transferand/or manipulation within harvesting chamber 40. Currently, a clearplastic adhesive tape commercially available from Hampton Research isused for sealing hanging drop experiments. This tape seals theequilibration wells while at the same time holding the hanging drop.This tape is compliant such that tape covering the crystal harvestingchambers 40 creates a minimal backpressure once fluid is pumped into thechannel. Should compliance present a problem, it is possible to providea narrow vent hole on the outlet side that is very hydrophobic, andtherefore would not let any liquid escape, only air.

[0082] The prototype crystallization chip in the PCG device wouldincorporate the vented air bellows design. This greatly simplifiestesting and makes it very user-friendly. For the device, a volume of 20μL can be used for each crystallization chamber; however, smallerchamber volumes of 10-100 nanoliters are readily possible. Threeapproaches can be used in the microfluidic circuit cartridges toinitiate protein crystallization and accompanying figures show theconceptual design for a single PCG experiment on the prototype board. Itshould be noted that all three approaches could be mixed and matchedonto a single board. The PCG techniques are: self-diffusion ofprecipitants and protein across a laminar boundary (see FIG. 7);turbulent mixing of all components—batch mode (see FIG. 8); and vaportransport into a desiccant or precipitant (see FIG. 9).

[0083] Referring now to FIG. 7, the interfacial diffusion approach willconsist of using 2×concentrations of protein and precipitant in eachfluid reservoir (volumes >10 μL) and each made up in 1× concentrationsof same buffer, salt and detergent. The two fluids are then injectedunder pressure in a 1:1 mixing ratio controlled by the diameter ofmicrochannels 42 into crystallization chamber 39. Chamber can be filledunder laminar flow conditions provided that it has at least onedimension of less than roughly 500 micrometers, and chamber is filledfairly slowly using gentle finger pressure (all other microfluidicstructures will be small enough to easily fulfill the requirements oflaminar flow). Pressure in the system is equilibrated by removing thefinger gently from vent hole 64. Air bellows 62 are then carefullysealed with clear adhesive tape in the same way as are fluid reservoirs32, 34 and harvesting chamber 40. Voiding of crystallization chamber 40into harvesting chamber 40 involves removal of the adhesive tapecovering harvesting chamber 39 and applying pressure on air bellows 62.

[0084] Referring now to FIG. 8, cartridge 50, which uses turbulentmixing for all components, operates in a batch mode. A turbulent mixingchamber 70 is inserted between fluid reservoirs 32, 34 andcrystallization chamber 39. Chamber 70 mixes the protein and precipitantfluids into a homogeneous liquid which is transported to chamber 39 forcrystallization. This design is particularly useful under microgravityconditions such as on a space shuttle mission, as the viscousprecipitants do not have time to mix and induce, nucleation during theduration of an extended mission.

[0085]FIG. 9 shows an example of cartridge 50 of FIG. 8 which uses theprinciples of vapor diffusion to operate. In this embodiment,crystallization chamber 39 is only partially filled after the fluids aremixed within mixing chamber 70. A predefined desiccant or precipitant 72is located within harvesting chamber 40 to absorb a fixing quantity ofsolution into chamber 40, increasing the concentration of protein withchamber 39, and inducing crystallization.

[0086] It would also be possible to take a starting protein solution anddialyze it against the appropriate starting fluid composition using anH-Filter prior to the crystallization experiment, should long termin-orbit storage in a particular buffer be deleterious to proteinstability. An H-filter setup could be incorporated into the design toeliminate irreversible protein aggregates. Referring now to FIG. 10,cartridge 50 contains fluid reservoir 32 filled with a protein sampleand fluid reservoir 34 containing a precipitant solution, as shown inthe previous examples. An additional fluid reservoir 80 is located oncartridge 50 which contains a protein and buffer solution. Allreservoirs are filled through inlets 52. Fluids from reservoirs 32, 80flow through microchannels 42 into a channel 32 which operates as anH-Filter to separate unwanted particles into a waste reservoir 84. Thefiltered solution travels through check valve 38 where it contacts fluidfrom reservoir 34 to form a laminar flow stream through crystallizationchamber 39. Another option is to just filter protein reservoir 32contents using a 0.22μ filter directly incorporated onto cartridge 50and placed just after the protein fluid reservoir 32 and before checkvalve 38. This type of filter is typically used to remove particulatematter for dynamic light scattering experiments.

[0087] Referring now to FIG. 11, a high density screeningcrystallization cartridge 50 is shown. Cartridge 50 contains fourcrystallization chambers 39. Chambers 39 have approximately a 0.5×0.5 mmcross-section. Protein solutions are added at a series of ports 86,while precipitant solutions are added at a series of ports 88. A seriesof valves 90 couple air bellows 62 to a series of filling chambers 92,which each correspond to a port 86. Each chamber 92 has a capacity of1-10 μl. A series of harvesting chambers 40 are each coupled to one ofchambers 39. Ports 88 are each connected to a fluid reservoir 34, whichin turn are coupled to a corresponding harvesting chamber 40. Each ofharvesting chambers 40 has a corresponding vent hole 94. Each harvestingchamber 40 has a capacity of approximately 50 μl, while each fluidreservoir has a capacity of between 0.1 and 0.5 ml.

[0088] In operation, ports 86 and 88 are filled with their respectivesolutions. With valves 90 in the closed position, mixing is achieved asthe solutions contact each other within chambers 39 to establish aconcentration gradient, as molecules diffuse across the interface zone,thus diluting the protein solution. Valves 90 are then openedindividually and the solutions are moved through chambers 39 under theforce provided by air bellows 62. During this protein crystallizationgrowth phase, vents 94 and 64, ports 86 and 88, and harvesting chambers40 are all sealed using adhesive tape. Harvesting occurs by openingvalves 90, which forces the contents of crystallization chambers 39 intoharvesting chambers 40.

[0089] Testing the design of the microfluidic crystallization chipsrequires the use of protein. Lysozyme and thaumatin PCG systems asinitial controls for evaluation of the performance of the chips andinstrumentation may be used in this embodiment.

[0090] A primary concern is the wetting of the fluid reservoirs toefficiently expel any air bubbles formed during the filling operation.This may be a question of having adequate pipette tips for liquidhandling and compatible fluid inlet dimensions. Siliconization may beused to control wetting. Rounded corners, oval or circular fluidreservoir shapes may be examined to minimize bubble entrapment.Dimension and placement of the vent hole should be studied as well aswhether filling should be done in a position where the cartridge isslanted to efficiently void air bubbles. All fluids are to be degassedprior to filling.

[0091] The shape of the crystallization chamber is important to ensurelaminar flow of the two liquids during its filling. A crystallizationchamber having a T-sensor structure should be sufficient for operation.However, for rapid inspection of PCG results, it is advantageous tolocalize PCG in a smaller region. Laminar flow in a crystallizationchamber can be readily monitored by injecting two fluids each containinga different colored dye.

[0092] The volume of the harvesting chamber should be of sufficient sizeto allow harvesting of the entire crystallization chamber contents aswell as addition of aliquots of mother liquor and cryo-protectantbuffer. The harvesting chamber shape should have rounded corners andallow facile access for crystal harvesting.

[0093] Plastic clear tape should be used for sealing the fluidreservoirs and harvesting chamber and tested for long-term stability andcompatibility with the microfluidic circuit cartridge. Attention shouldbe paid in PCG trials to ease of peeling off the tape from the circuitboards. It may also be advantageous for efficient handling to provide abacking to the plastic clear adhesive tape that peels off exposing theadherent surface for subsequent sealing. Visual cues can be provided onthe circuit cartridges of where to place the sealing tape.

[0094] The microfluidic cards are made of plastic laminates bondedtogether with adhesive. The plastic laminate composition is mylar, whichis a very resistant material. The fluid compatibility and long-termfluid integrity, however, needs to be assessed and is addressed in thework packages. Problems with PCG fluid compatibility are not anticipatedwith either the laminate adhesive and mylar. Alternatively, glass orsilicon can be used if the fluid incompatibility is severe. Under thesecircumstances, it should be possible to perform all fluidic developmentusing the laminate method; however, when it comes to mass production, itmay be desirable to make the structures out of glass or silicon.

[0095] The microfluidic integrated circuit cartridges, when sealed withthe covering adhesive film, comprise one level of fluid containment. Thefluid driver interface connection on the circuit cartridges is airtight,while the air bellows design does not compromise the containment level.Fifty (50) microfluidic integrated circuit cards containing up to 20individual PCG experiments each or 1000 PCG experiments in all could fitwith external controllers into a sealed container within the volume of amid-deck locker that provides the second level of containment and, ifrequired, temperature control.

[0096] Usually, microfluidic systems require some kind of fluidic driverto operate, e.g., piezoelectric pumps, micro-syringe pumps,electroosmotic pumps, etc. In two previous patent applications, U.S.patent application Ser. No. 09/415,404 and U.S. patent application Ser.No. 60/189,163, which applications are hereby incorporated by reference,there are shown microfluidic systems that are entirely driven by aninherently available force such as gravity, capillary action, absorptionin porous materials, chemically induced pressures or vacuums (e.g., by areaction of water with a drying agent), or by vacuum or pressuregenerated by simple manual action. Such devices are extremely simple andcheap, do not require electricity, can be manufactured, for example,entirely out of a single material such as plastic, with a method such asinjection molding, and are simple to operate.

[0097] One embodiment of a device according to the present inventionwould comprise a hydrostatic pressure-driven cartridge, in which thehydrostatic pressure heads are manufactured as part of the cartridgeitself. The cartridge would then be placed on its side so that thegravity pulls the liquids through the channels.

[0098] Another embodiment comprises a cartridge on which air spacesunder a flexible membrane are in fluid connection with the microfluidicfluid circuit. These compressible airspaces can then be used to aspirateliquids into the channels, or to apply pressure to push liquids tovarious points on the cartridge, for example, to prime a microfluidiccircuit or to siphon fluids until it starts working by gravitationalforce.

[0099] Another embodiment contains chambers in which certain chemicalliquids (e.g., ethanol, butane, carbon dioxide, organic solvents, etc.or any substance which has a partial pressure at operating temperaturethat generates enough force to push liquids through a microfluidicsystem at desired flow rates) are present in equilibrium with theirgaseous phases. These spaces are in fluid connection with parts of themicrofluidic circuit and the other reagents, and the pressure in thesechambers push the reagents and samples through the channels of themicrofluidic circuit.

[0100] In addition to filling by gravity or syringe, bellows-drivenmicrofluidic structures have been manufactured in which the bellows areintegrated into the laminate as either aspiration or pressurizationbubbles. Vents can be placed at various places on the cartridges toallow directional flow of the fluids.

[0101] It is also possible to prefill cartridges during manufacturing. Apredefined volume of fluid can be placed on a reservoir on an openlaminate, which is then sealed with tape, or a cover layer. This actioncan also be used to drive the fluid to where it should be inside themicrofluidic circuit.

[0102] While the present invention has been shown and described in termsof a preferred embodiment thereof, it will be understood that thisinvention is not limited to this particular embodiment and that manychanges and modifications may be made without departing from the truespirit and scope of the invention as defined in the appended claims.

What is claimed is: 1) A device for promoting protein crystallizationgrowth from solution, comprising: a body structure; means located withinsaid body structure for introduction of at least one solution containingprotein and at least one solution containing a solvent; and at least onemicrofluidic channel connected to said introduction means wherein saidprotein solution and said solvent solution interact to induce formationof protein crystals within said channel. 2) The device of claim 1,wherein said protein solution and said solvent solution flow laminarlyin parallel contact within said microfluidic channel to establish aconcentration gradient within said channel, allowing for proteincrystallization. 3) The device of claim 1, wherein said protein solutionintroduction means and said solvent solution introduction means are eachconnected to said crystallization channel by a microfluidic channel. 4)The device of claim 3, wherein said protein microfluidic channel, saidsolvent microfluidic channel, and said crystallization channel form aT-Sensor structure. 5) The device of claim 1, further comprising achamber coupled to said crystallization channel for harvesting saidformed protein crystals. 6) The device of claim 1, further comprisingfluid movement generating means coupled to said protein solutionintroduction means and said solvent solution introduction means forpropelling said solutions through said crystallization channel. 7) Thedevice of claim 6, wherein said fluid movement generating meanscomprises an air bellows. 8) The device of claim 1, further comprising amixing means, coupled between said protein solution introduction meansand said solvent introduction means interface and said crystallizationchannel for mixing said protein solution and said solvent solutioncompletely to form a homogeneous mixture. 9) The device of claim 8,wherein said mixing means comprises a jet vortex mixer. 10) The deviceof claim 9, further comprising a solvent absorbing means coupled to saidcrystallization channel for absorbing solvent from said homogeneousmixture to increase the concentration of protein within said mixture,thereby inducing increased protein crystallization. 11) A device forpromoting protein crystallization growth from solution, comprising: abody structure; means located within said body structure forintroduction of at least one solution containing protein, at least onesolution containing a solvent, and at least one solution containing acombination of protein and a buffer; a microfluidic structure coupled tosaid protein solvent introduction means and said combination protein andbuffer solution introduction means for flowing said solutions laminarlyin parallel to remove irreversible protein aggregates from said combinedsolutions; and at least one microfluidic channel connected to saidsolvent introduction means and the output of said microfluidic structurewherein said solvent solution and said combined solutions interact toinduce formation of protein crystals within said channel. 12) The deviceof claim 11, further comprising a waste chamber coupled to saidmicrofluidic structure to retain said irreversible protein aggregates.13) The device of claim 11, further comprising a chamber coupled to saidcrystallization chamber for harvesting said formed protein crystals. 14)The device of claim 11, further comprising fluid movement generatingmeans coupled to said protein solution introduction means, said solventsolution introduction means, and said combined protein and buffersolution introduction means for propelling said solutions through saidcrystallization channel. 15) The device of claim 14, wherein said fluidmovement generating means comprises an air pump. 16) The device of claim11, wherein said microfluidic structure comprises an H-filter. 17) Thedevice of claim 1, wherein said body structure is constructed fromplastic.